A rat histone H2B pseudogene is closely associated with the histone H1d gene.

A 9 kb EcoRI restriction fragment was isolated from a recombinant phage out of a rat genomic library. This DNA fragment contains a rat H1d histone gene, its flanking sequences and a H2B histone pseudogene closely associated with the H1d gene. A comparison of the H2B pseudogene with human H2B genes flanking regions reveals sequence homologies to a human H2B histone gene (Albig, W. et al. (1991) Genomics 10, 940-948).

Expression of murine H1 histone genes during postnatal development.

Murine genes encoding the seven H1 histone isoforms H1.1-H1.5, H1(o) and H1t have been isolated and sequenced. We have established expression patterns of these genes in several tissues during postnatal development. For that analysis, RNase protection assay rather than Northern blot hybridization was used, since the sequences of these genes are highly similar and would cross-hybridize under Northern blot conditions. Expression patterns of H1.1 to H1.5 and H1(o) were determined in tissues of animals at days 5, 9 and 20 after birth and of adult mice. In addition, RNA was analyzed in three mouse cell lines (NIH3T3, P19, TM4). Transcription of the subtype genes H1.2 and H1.4 was found in all tissues and cell lines studied. The most varied expression patterns were obtained with the H1.1 subtype. H1.1 mRNA was found at high concentrations in thymus and spleen throughout development and in testis beginning with a low expression in 5-day-old animals and increasing levels in testis RNA from 9- and 20-day-old and adult mice. H1(o) mRNA was found primarily in highly differentiated tissues with concentrations decreasing from 5-day-old to adult animals.

The expression of human H2A-H2B histone gene pairs is regulated by multiple sequence elements in their joint promoters.

The majority of human H2A and H2B histone genes are organized as gene pairs: 14 H2A-H2B gene pairs, one solitary H2A gene and three solitary H2B genes have been described. Two of the H2A genes and two of the H2B genes arranged within gene pairs are pseudogenes. The gene pairs are organized with divergent transcriptional orientation, and the coding regions of the respective H2A and H2B genes are separated by about 320 nucleotide pairs that form overlapping promoter regions. Comparison of promoters of H2A-H2B gene pairs has previously shown that these belong to two different groups (groups I and II) which are characterized by specific patterns of conserved sequence elements. We have constructed a reporter gene vector that allows the simultaneous analysis of both genes regulated by the divergent promoters belonging to group I or II, respectively. Firefly-luciferase and beta-galactosidase genes were taken as reporter genes. Site directed mutagenesis performed at individual promoter elements revealed that individual sequence elements within both groups of promoters functionally depend on each other and may contribute to a coordinate expression of paired H2A and H2B genes through assembly of their joint promoter into a mutually dependent promoter complex. Group II promoters are characterized by the presence of an E2F binding site upstream of the H2A gene-proximal TATA box. Immediately upstream of the E2F element, we have identified a highly conserved octanucleotide CACAGCTT (RT-1) that exists in all human group II H2A-H2B gene promoters. Protein binding studies at the RT-1 element indicate factor binding to this sequence. Site directed mutagenesis indicates that both the E2F element and the RT-1 motif are essential for full promoter activity.

Structure and expression of the mouse testicular H1 histone gene (H1t).

A mouse genomic library was screened with a human testicular H1 (H1t) gene fragment. One phage containing the testis specific mouse H1t histone gene and its flanking regions was isolated. Northern blot analysis showed that the mouse H1t gene is expressed only in mouse testis at the stage of pachytene spermatocytes and that the H1t mRNA is not polyadenylated. This mouse H1t gene encodes a protein which differs from the somatic mouse H1 proteins, but is similar to the known H1t proteins from rat, and man.

Selective expression of specific histone H4 genes reflects distinctions in transcription factor interactions with divergent H4 promoter elements.

Expression of many histone H4 genes is stringently controlled during the cell cycle to maintain a functional coupling of histone biosynthesis with DNA replication. The histone H4 multigene family provides a paradigm for understanding cell cycle control of gene transcription. All functional histone H4 gene copies are highly conserved in the mRNA coding region. However, the putative promoter regions of these H4 genes are divergent. We analyzed three representative mouse H4 genes to assess whether variation in H4 promoter sequences has functional consequences for the relative level and temporal control of expression of distinct H4 genes. Using S1 nuclease protection assays with gene-specific probes and RNA from synchronized cells, we show that the mRNA level of each H4 gene is temporally coupled to DNA synthesis. However, there are differences in the relative mRNA levels of these three H4 gene copies in several cell types. Based on gel shift assays, nucleotide variations in the promoters of these H4 genes preclude or reduce binding of several histone gene transcription factors, including IRF2, HiNF-D, SP-1 and/or YY1. Therefore, differential regulation of H4 genes is directly attributable to evolutionary divergence in H4 promoter organization which dictates the potential for regulatory interactions with cognate H4 transcription factors. This regulatory flexibility in H4 promoter organization may maximize options for transcriptional control of histone H4 gene expression in response to the onset of DNA synthesis and cell cycle progression in a broad spectrum of cell types and developmental stages.

The telomeric region is excluded from nucleosomal fragmentation during apoptosis, but the bulk nuclear chromatin is randomly degraded.

Characteristic steps during cellular apoptosis are the induction of chromatin condensation and subsequent DNA fragmentation, finally leading to the formation of oligomers of nucleosomes. We have examined the kinetics and local distribution of this nucleosomal fragmentation within different genomic regions. For the induction of apoptosis, HL60 cells were treated with the water-soluble camptothecin derivative topotecan (a topoisomerase I inhibitor). The genomic origin of the fragments was analysed by Southern blot hybridisation of the cleaved DNA. In these experiments we observed similar hybridisation patterns of the fragmented DNA, indicating a random and synchronous cleavage of the nuclear chromatin. However, hybridisation with a telomeric probe revealed that, in contrast to the other analysed genomic regions, the telomeric chromatin was not cleaved into nucleosomal fragments despite our observation that the telomeric DNA in HL60 cells is organised in nucleosomes. We determined just a minor shortening of the telomeric repeats early during apoptosis. These observations suggest that telomeric chromatin is excluded from internucleosomal cleavage during apoptosis.

Differential distribution of lysine and arginine residues in the closely related histones H1 and H5. Analysis of a human H1 gene.

A human H1 histone gene and its flanking sequences were isolated from a human gene library using a fragment of the duck H5 histone gene as a hybridization probe. The primary structure of this human H1 histone (as deduced from the nucleotide sequence of the gene) reveals a close homology of H1 and H5 histones and fits the three-domain organization of all members of the H1 histone family. Within this protein organization, the C-terminal domain of H1 differs from the arginine-rich H5 in its distribution of the basic amino acids: the C-terminal domain of the human H1 shows only one arginine and most of the H5 specific arginine positions show lysine instead.

Conserved dyad symmetry structures at the 3' end of H5 histone genes. Analysis of the duck H5 gene.

The duck H5 histone gene and its flanking DNA have been isolated and sequenced. S1 nuclease mapping reveals that transcription starts 149 nucleotides upstream of the initiation codon and that the site of polyadenylation is located 200 nucleotides downstream of the termination codon. A comparison with the chicken H5 gene demonstrates that the 3' non-translated segment of the polyadenylated H5 mRNA carries two conserved dyad symmetry sequences. The first potential hairpin is located directly after the termination codon of the H5 gene and is highly conserved, whereas the second stem and loop structure maps shortly upstream of the polyadenylation site and shows a homology block at the central part of this inverted DNA repeat.

Organization and expression of the developmentally regulated H1(o) histone gene in vertebrates.

The H1 class of histones comprises several main-type, S-phase dependent isoforms and, in addition, a sperm-specific H1t and a peculiar subtype, H1 zero, which is confined to highly differentiated cells. In contrast to main type histone genes, the H1 zero gene expression does not strictly depend on DNA replication. Also in contrast to the other H1 subtype genes, the mammalian H1 zero gene is not included in a histone gene cluster and its mRNA differs in structure, size and mode of processing from other histone mRNAs. The regulation of expression of the H1 zero gene varies from main type H1 genes in several respects. This is manifested in the promoter structure which contains sequence elements that are also found in main type H1 promoters, but also shows regulatory motifs which appear to be involved in a developmental regulation of the H1 zero gene, such as a retinoic acid receptor binding site, which has been described in the mammalian H1 zero gene promoter.

Core histones and linker histones are imported into the nucleus by different pathways.

Histones are the major structural proteins in eukaryotic chromosomes. This group of small very basic proteins consists of the H1 linker histones and the core histones H2A, H2B, H3 and H4. Despite their small size, the nuclear import of histones occurs by an active transport mechanism and not simply by diffusion. Histones contain several nuclear localisation signals (NLS) that can be subdivided into two different types of signal structures. We have previously shown that H1 histones are transported by a heterodimeric import receptor complex consisting of importin beta and importin 7, and we now describe the receptors required for the import of the core histones. Competition experiments using the in vitro transport assay indicate that the import pathway of the core histones differs from that of the linker histones and of nuclear proteins with classical NLS. In vitro binding assays show that each of the import receptors importin beta, importin 5, importin 7 and transportin, has the capacity to bind to any of the four core histones. Reconstitution experiments with recombinant factors indicate that each of these factors can independently serve as an import receptor for each of the core histones.

Human H1 histones: conserved and varied sequence elements in two H1 subtype genes.

The genes coding for two different human H1 histones were isolated, and the primary structures were deduced from the nucleotide sequences. The genes differ from each other and from any other vertebrate H1 structure described until now. The differences occur mainly within the N- and C-terminal H1 domains, whereas the central part of the protein is highly conserved. Within the flanking domains, however, some sequence elements are shared by different H1 subtype genes. An octapeptide, which has been described in C-terminal domains of most H1 histones, is found in both H1 subtypes. The nucleotide sequences of the flanking portions of both H1 genes show conserved motifs at established regulatory sites, but otherwise these 3' and 5' noncoding sequences of both genes differ substantially.

Structure of a duck H3 variant histone gene: a H3 subtype with four cysteine residues.

A duck recombinant DNA phage library was screened for H3 histone genes, and the sequence of a variant H3 gene, which appears not to be part of a histone gene cluster, has been determined. As derived from the nucleotide sequence, this gene codes for a 135-amino acid (aa) protein (as any other H3) and shows 10 aa substitutions compared with most published H3 structures. Six of these aa changes are based on one nucleotide (nt) substitutions in arginine codons. This results in three new histidines and, in addition to the highly conserved cysteine at position 110, three more cysteines are found in this H3 histone subtype.

Structure and expression of the human gene encoding testicular H1 histone (H1t).

The gene coding for the human H1t histone, a testis-specific H1 subtype, was isolated from a genomic library using a human somatic H1 gene as a hybridization probe. The corresponding mRNA is not polyadenylated and encodes a 206-amino-acid protein. Sequence analysis and S1 nuclease mapping of the human H1t gene reveals that the 5' flanking region contains several consensus promoter elements, as described for somatic, i.e., S-phase-dependent H1 subtype genes. The 3' region includes the stem-and-loop structure necessary for mRNA processing of most histone mRNAs. Northern blot analysis with RNAs from different human tissues and cell lines revealed that only testicular RNA hybridized with this gene probe.

Characterization of the H1.5 gene completes the set of human H1 subtype genes.

The H1 histone family in mammals contains at least seven subtypes. In the past we have isolated six of the seven genes encoding these isoforms. To complete the set of the human H1 histone genes, we have designed two PCR primers deduced from a partially published sequence of the remaining histone H1 gene [Carozzi et al. (1984) Science 224, 1115-1118] and from a consensus sequence which we have derived from the conserved region of human histone H1 genes. Using these primers we have amplified a 417-bp DNA fragment from total human DNA. This fragment was used for screening a human phage genomic library. Two overlapping clones were isolated. The region contains a set of 5 genes representing each of the five histone classes. In continuation of our numbering of human H1 genes, we have named this H1 gene H1.5. This gene encodes a protein almost identical to the previously published protein sequence designated H1a [Ohe et al. (1986) J. Biochem. 100, 359-368]; since the changes are in a region of some uncertainty of the peptide sequencing, we conclude that the newly isolated gene codes for the H1a protein. The structures of the flanking regions of the genes except the H2B gene are typical for histone genes. They include: (1) a CCAAT element in the promotor region, (2) a TATA box and (3) a palindromic termination element. The H2B sequence shows no typical regulatory elements and no complete ORF, therefore we consider it as a pseudogene. The expression of the H1.5 gene was examined in several cell lines.

Characterization of the two H1(zero)-encoding genes from Xenopus laevis.

We have analyzed the promoter and the coding sequences of the two homologous histone H1(zero)-encoding genes from Xenopus laevis, here termed H1(zero)-1 and H1(zero)-2. Both genes encode proteins of 193 amino acids and differ at just 16 amino-acid residues. Putative regulatory sequences identified in the promoter region are the same and are highly conserved. However, significant differences exist in the 5' untranslated regions (UTR) of the transcribed sequences of these two genes, such as several deletions in the 5'-UTR of the H1(zero)-2 gene in comparison with the H1(zero)-1 gene 5'-UTR. The 3'-UTR is a short sequence of about 200 bp which is unexpected compared with the long 3'-UTR of mammalian H1(zero) mRNA, but it is in the same size range as in avian H5 mRNA. Thus, the main differences between these two genes are observed in sequences potentially involved in the regulation of the H1(zero) gene expression such as the 5'-UTR. The two genes are expressed during embryogenesis and in several adult tissues. We discuss these findings in terms of the evolution of histone H1(zero) genes in vertebrates and the appearance of histone H5 in avian species.

Transcriptional regulation of the human replacement histone gene H3.3B.

In contrast to the cell-cycle-dependent histone genes, replacement histone genes are transcribed independently of DNA replication and their expression is upregulated during differentiation. We have investigated the transcriptional regulation of the recently characterized human replacement histone gene H3.3B. Using reporter gene assays of promoter-luciferase gene-constructs, we show that promoter activity largely depends on an intact Oct and CRE/TRE element within the proximal 145 bp of the promoter. DNase I footprinting revealed binding of proteins to a 40-bp region covering these two elements. Band shift experiments identified binding proteins as Oct-1 and factors of the CREB/ATF and AP-1 family, respectively. The unexpected transcriptional regulation of this replacement histone gene is discussed.

Heterologous expression of human H1 histones in yeast.

The complete set of seven human H1 histone subtype genes was heterologously expressed in yeast. Since Saccharomyces cerevisiae lacks standard histone H1 we could isolate each recombinantly expressed human H1 subtype in pure form without contamination by endogenous H I histones. For isolation of the H1 histones in this expression system no tagging was needed and the isoforms could be extracted with the authentic primary structure by a single extraction step with 5%(0.74 M) perchloric acid. The isolated H1 histone proteins were used to assign the subtype genes to the corresponding protein spots or peaks after two-dimensional gel electrophoresis and capillary zone electrophoresis, respectively. This allowed us to correlate transcriptional data with protein data, which was barely possible until now.

Effects of antineoplastic phospholipids on parameters of cell differentiation in U937 cells.

The proliferation of the human promonocytic leukemia cell line U937 is inhibited by several ether lipids, ether lipid analogues and by phorbol esters. An early effect of this retardation of cell growth is the induction of a basic chromosomal protein, histone H1(0). Northern blot analysis of H1(0) mRNA levels reveals an increase of the mRNA concentration within a few hours after addition of hexadecylphosphocholine and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine. This early effect on the synthesis of a subtype of H1 proteins precedes the expression of several parameters of the monocytic differentiation of U937 cells.

Varied expression patterns of human H1 histone genes in different cell lines.

Five main type H1 histones have been described in man (H1.1-H1.5) in addition to the testis specific type H1t and the replacement subtype H1 degrees, which is found mainly in highly differentiated cells. We have isolated this whole complement of H1 genes and have studied the expression of the seven human H1 subtype genes in several cell lines. The RNAase protection assay was used to discriminate between the very similar transcripts derived from the seven H1 subtype genes. With the exception of H1.2 and H1.4, we found substantial differences between the H1 mRNA levels in the different cell lines tested. No H1.1 mRNA was detected in most of the cell lines and just a low level of H1.1 mRNA was found in human testis. In contrast to the differential patterns of the other subtypes, H1.2 and H1.4 were in all cells expressed at a high level, indicating a basal function compared with the other H1 histones. Because differences in the timing of H1 protein subtype synthesis have been reported, we have analyzed the kinetics of accumulation of H1 subtypes in synchronized HeLa cells and observed that all H1 subtypes examined (H1 degrees, H1.2-H1.5) were expressed in a replication-dependent manner. The analysis showed a differential rise of mRNA levels during S-phase, from four-fold (H1 degrees) to 15-fold (H1.5). Our results may point at a specific function of each subtype and suggest that expression of the H1 histone subtype genes depends on common S-phase-depent factors as well as on individual regulatory systems. Thus, the data presented here provide a basis for further analysis of the regulation and function of the complex H1 gene and protein family.

Association of histone H4 genes with the mammalian testis-specific H1t histone gene.

Mouse and human H4 genes associated with the testis-specific H1t gene were isolated from genomic libraries and were sequenced. The deduced amino acid sequences are identical to other mouse or human H4 histones, but the genes differ significantly in their nucleotide sequences. Both the human and the mouse genes are located on the same DNA strand compared with the H1t gene. In contrast to this identical transcriptional orientation of H1t and its neighboring H4 gene in mouse and man, an H4 gene with the opposite orientation has been described in the vicinity of the rat H1t gene. Northern blot analysis of RNA from testicular cells separated by centrifugal elutriation, S1 nuclease mapping, and reverse transcriptase polymerase chain reaction (RT-PCR) amplification show that both the murine and human H4 genes, like the H1t gene, are expressed in testicular cells, whereas the H4 genes, in contrast to the H1t gene, are expressed in nontesticular human and mouse cell culture cells.